Olive-growing represents a crucial production industry for the entire Mediterranean area in general and Italy in particular. In Italy olive trees are distributed on mainly hilly surfaces and represent about a fifth of the surface designated for olive trees worldwide. However, this industry suffers the drawback of considerable generation of wastes which in particular comprise vegetable material (leaves and prunnings) and actual olive oil production residues (VW, wet and used pomaces). The latter are characterised by a strong pollution load, and hence require suitable procedures in order to be disposed according to relevant environmental laws (e.g., Italian Law DL 574 dated Nov. 11, 1996). Over roughly the last ten years the scientific and industrial community has increased its efforts to provide a solution to the problem regarding disposal of olive oil wastewaters. Among the proposed solutions, are those aimed at creating value by recovering useful substances from wastewaters, particularly polyphenol substances with which numerous biomedical and functional properties are associated.
Compounds of vegetable origin are capable of expressing different biological properties (e.g. anti-oxidant, anti-radical, antimicrobial properties). In many cases, the single molecule is less active with respect to the mixture of the compounds, thus suggesting synergistic action among them. In fact, raw extracts of vegetable origin often reveal greater bioactivity with respect to that observable when using the simple compounds present therein. Antiradical activity of vegetable extracts is measured by means of an in vitro test using the DPPH (diphenylpicrylhydrazyl) stable radical which simulates the activity of such extracts against endogenous oxygenated radicals, such as hydroxide and superoxide radicals. The use of the DPPH radical allows for the analysis of radical reduction kinetics and also for calculating the EC50, i.e. the concentration of extract that reduces 50% of the radical.
Hydroxytyrosol and oleuropein aglycone are potent antioxidant and cardioprotective agents. Oleuropein reveals coronary-dilating, hypoglycemic and anticholesterolemic activity. Similar to hydroxytyrosol, it delays oxidation of LDLs (Low Density Lipoprotein). Hydroxytyrosol has been shown to reduce the gene expression of iNOS and COX-2 cell lines, thus preventing the activation of the NF-KB transcription factors, key factors in atherosclerosis, STAT-1α and IRF-1. Prevention of the activation of NF-KB was also observed in an ex-vivo study of monocytes of healthy volunteers exposed to consumption of olive oil, suggesting possible anti-inflammatory effects of the oil. There are numerous studies regarding the biological activities of molecules with a phenylpropanoid structure, demonstrating an antioxidant, anti-inflammatory, antiviral and antifungal activity thereof. Such studies are mainly focused on evaluating the effects of verbascoside or acteoside, one of the most studied phenylpropanoids to date. Verbascoside also revealed in vitro immunomodulatory activity, in particular by increasing the chemotactic activity of neutrophils. Verbascoside demonstrated, on PC12 neuronal cell culture, protection activity against neurotoxicity induced by the 1-methyl-4-phenylpyridine (MPP+) ion, an ion which, activating caspase-3, causes serious oxidative stress in cells. Antineoplastic activities have also been suggested for both verbascoside and the isomer thereof, isoacteoside. In vivo tests on P-388 murine leukaemia cells, revealed that the two phenylpropanoids showed an ED50 cytotoxic action equivalent to 10 μg/mL for isoacteoside and 26 μg/mL regarding verbascoside. A further polyphenol subclass (more specifically, a flavonoid subclass) of a particular biological interest, specifically present in the pulps of pigmented olives, is the class of anthocyanoside compounds or anthocyanines consisting, in particular, of glycoside derivatives of cyanidine, the most abundant of them being cyanidine-3-O-rutinoside. Anthocyanosides are pigments specifically present in the skin of grapes and small fruits, marketed as standardized extracts of berries or marc residues, and widely studied because of their biological activity. The term anthocyanidines, referred to the class of the corresponding non-glycosylated compounds (cyanidine being one of the major representatives thereof), has been created to designate the substances responsible for the colour of flowers, and is relevant to a group of water-soluble pigments responsible for the colours red, pink, violet and blue of most flowers and fruits. The drugs containing anthocyanidines were used in galenic preparations for the treatment of the symptomatology connected with capillary fragility. Such compounds also show high antioxidant activity and are capable of protecting cells from oxidative damages caused by free radicals.
US2002198415 (A1), US2008090000(A1), US2010216874(A1) describe, starting from olive oil production industry wastes, how to obtain polyphenol-based extracts, through acid treatment of the VW and a prolonged storage thereof up to 12 months at a pH between 1 and 6 with the aim of determining the conversion of oleuropein into hydroxytyrosol. After incubation, the initial oleuropein was converted (about 75-90%) into hydroxytyrosol. WO2007/013032 describes a process for recovering a concentrate rich in hydroxytyrosol from residues of the olive oil production industry, particularly vegetation waters and pruning residues (leaves). Said process provides for the use, after extracting using a solvent (water or alcohol), of extraction systems with supercritical fluids, nanofiltration or, alternatively, reverse osmosis for recovering hydroxytyrosol and minor polar compounds. The product thus obtained is an hydroxytyrosol-based extract. WO2005/123603 describes a separation process based on membrane technologies specifically aimed at recovering compounds of interest from the VW. In such process, to the various tangential membrane filtration separation operations, there was introduced an initial filtration aimed at maximising the commercial useful polyphenol (such as hydroxytyrosol) content to the detriment of the possible oleuropein still present in the VW. Pre-treatment consists in acidifying still fresh VW to a pH of about 3-4.5, followed by enzymatic hydrolysis. After separating the liquid product thus treated by centrifuge, there follows a series of cascade tangential filtration operations, which includes microfiltration followed by nanofiltration and lastly by a reverse osmosis process, obtaining from the various retentates polyphenol fractions with different degrees of purification and from the reverse osmosis permeate purified water that can be used for producing beverages. WO2008/090460 describes a further example of a process for recovering the hydroxytyrosol component from the olive oil production industry residues, in which milling wastes are not used alone but rather also include a given amount of green olives with the aim of obtaining a product particularly enriched in hydroxytyrosol. This reference proposes a first acid hydrolysis treatment at a temperature greater than the reflux temperature for the initial material (pomaces and green olive pulps), followed by clarification of the resulting product (for example by filtration), in turn followed by a treatment on an ion exchange chromatography resin. The product adsorbed on such column, after elution, in turn may be supplied to a second chromatography column loaded with a non-ionic adsorbent resin. The product adsorbed on the latter resin, after elution, is further concentrated in hydroxytyrosol, if necessary, through a membrane tangential filtration, specifically reverse osmosis, in which retentate is the desired product. WO2009/016482 describes a process for the treatment of vegetable matter, including the olive VW. The authors describe a process comprising acidification, two steps of enzymatic lysis, providing for a passage for the separation of the solids between the two enzymatic lysis steps, microfiltration and vacuum evaporation.